Malaria, a devastating disease caused by the human protozoan parasite Plasmodium falciparum, affects 300-500 million people annually. Resistance to mainstay anti-malarial drugs is a challenge facing disease control efforts. Proteases are an attractive avenue for development of anti-malarials since they play crucial roles in P. falciparum biology. Studies using general inhibitors have confirmed a role for serine proteases in invasion and rupture. However, the association of specific proteases with these processes has been difficult due to the lack of facile genetic manipulations of P. falciparum and the lack of specific reagents to inhibit protease targets. We will take advantage of a library comprising of the highly class specific reactivity of several serine protease inhibitor scaffolds that form covalent intermediates that we have accumulated to identify new serine protease inhibitors. This library of compounds will be screened using a flow cytometry- based phenotypic assay in which parasite growth, replication, rupture and invasion can be monitored (Aim 1). The small molecule compounds identified in this forward chemical genetic approach will be developed into activity based probes (ABP) to identify the target proteases and used to further dissect the functional roles of the target proteases (Aim 2). The results from this proposed study should facilitate the assigning of functions to proteases that play critical roles in P. falciparum life cycle. [unreadable] [unreadable] [unreadable]